The Identification of Potential Factors Associated with the Development of Type 2 Diabetes
2008; Elsevier BV; Volume: 7; Issue: 8 Linguagem: Inglês
10.1074/mcp.m700478-mcp200
ISSN1535-9484
AutoresHongfang Lu, Ying Yang, Emma M. Allister, Nadeeja Wijesekara, Michael B. Wheeler,
Tópico(s)Metabolism, Diabetes, and Cancer
ResumoType 2 diabetes (T2D) arises when pancreatic β-cells fail to compensate for systemic insulin resistance with appropriate insulin secretion. However, the link between insulin resistance and β-cell failure in T2D is not fully understood. To explore this association, we studied transgenic MKR mice that initially develop insulin resistance in skeletal muscle but by 8 weeks of age have T2D. In the present study, global islet protein and gene expression changes were characterized in diabetic MKR versus non-diabetic control mice at 10 weeks of age. Using a quantitative proteomics approach (isobaric tags for relative and absolute quantification (iTRAQ)), 159 proteins were differentially expressed in MKR compared with control islets. Marked up-regulation of protein biosynthesis and endoplasmic reticulum stress pathways and parallel down-regulation in insulin processing/secretion, energy utilization, and metabolism were observed. A fraction of the differentially expressed proteins identified (including GLUT2, DNAJC3, VAMP2, RAB3A, and PC1/3) were linked previously to insulin-secretory defects and T2D. However, many proteins for the first time were associated with islet dysfunction, including the unfolded protein response proteins (ERP72, ERP44, ERP29, PPIB, FKBP2, FKBP11, and DNAJB11), endoplasmic reticulum-associated degradation proteins (VCP and UFM1), and multiple proteins associated with mitochondrial energy metabolism (NDUFA9, UQCRH, COX2, COX4I1, COX5A, ATP6V1B2, ATP6V1H, ANT1, ANT2, ETFA, and ETFB). The mRNA expression level corresponding to these proteins was examined by microarray, and then a small subset was validated using quantitative real time PCR and Western blot analyses. Importantly ∼54% of differentially expressed proteins in MKR islets (including proteins involved in proinsulin processing, protein biosynthesis, and mitochondrial oxidation) showed changes in the proteome but not transcriptome, suggesting post-transcriptional regulation. These results underscore the importance of integrated mRNA and protein expression measurements and validate the use of the iTRAQ method combined with microarray to assess global protein and gene changes involved in the development of T2D. Type 2 diabetes (T2D) arises when pancreatic β-cells fail to compensate for systemic insulin resistance with appropriate insulin secretion. However, the link between insulin resistance and β-cell failure in T2D is not fully understood. To explore this association, we studied transgenic MKR mice that initially develop insulin resistance in skeletal muscle but by 8 weeks of age have T2D. In the present study, global islet protein and gene expression changes were characterized in diabetic MKR versus non-diabetic control mice at 10 weeks of age. Using a quantitative proteomics approach (isobaric tags for relative and absolute quantification (iTRAQ)), 159 proteins were differentially expressed in MKR compared with control islets. Marked up-regulation of protein biosynthesis and endoplasmic reticulum stress pathways and parallel down-regulation in insulin processing/secretion, energy utilization, and metabolism were observed. A fraction of the differentially expressed proteins identified (including GLUT2, DNAJC3, VAMP2, RAB3A, and PC1/3) were linked previously to insulin-secretory defects and T2D. However, many proteins for the first time were associated with islet dysfunction, including the unfolded protein response proteins (ERP72, ERP44, ERP29, PPIB, FKBP2, FKBP11, and DNAJB11), endoplasmic reticulum-associated degradation proteins (VCP and UFM1), and multiple proteins associated with mitochondrial energy metabolism (NDUFA9, UQCRH, COX2, COX4I1, COX5A, ATP6V1B2, ATP6V1H, ANT1, ANT2, ETFA, and ETFB). The mRNA expression level corresponding to these proteins was examined by microarray, and then a small subset was validated using quantitative real time PCR and Western blot analyses. Importantly ∼54% of differentially expressed proteins in MKR islets (including proteins involved in proinsulin processing, protein biosynthesis, and mitochondrial oxidation) showed changes in the proteome but not transcriptome, suggesting post-transcriptional regulation. These results underscore the importance of integrated mRNA and protein expression measurements and validate the use of the iTRAQ method combined with microarray to assess global protein and gene changes involved in the development of T2D. The prevalence of type 2 diabetes (T2D) 1The abbreviations used are: T2D, type 2 diabetes; ER, endoplasmic reticulum; ERAD, ER-associated degradation; FFA, free fatty acid; GSIS, glucose-stimulated insulin secretion; iTRAQ, isobaric tags for relative and absolute quantification; MKR mouse, a transgenic mouse with a dominant-negative insulin-like growth factor-I receptor (KR-IGF-IR) specifically targeted to the skeletal muscle; qPCR, quantitative real time PCR; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; UPR, unfolded protein response; WT, wild-type; EF, error factor; PDI, protein-disulfide isomerase; GCOS, Affymetrix GeneChip Operating Software; ID, identity; EIF, eukaryotic initiation factor; UFM1, ubiquitin-fold modifier 1; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; ISG, insulin secretory granule; SYTL4, synaptotagmin-like protein-4/granuphilin; PCX, pyruvate carboxylase; ETF, electron transfer flavin protein; PPIB, peptidyl-prolyl cis-trans isomerase B; VCP, valosin-containing protein; CPE, carboxypeptidase E; UQCRH, ubiquinol-cytochrome c reductase complex; EGF, epidermal growth factor; ARMET, Arginine-rich, mutated in early stage tumors. 1The abbreviations used are: T2D, type 2 diabetes; ER, endoplasmic reticulum; ERAD, ER-associated degradation; FFA, free fatty acid; GSIS, glucose-stimulated insulin secretion; iTRAQ, isobaric tags for relative and absolute quantification; MKR mouse, a transgenic mouse with a dominant-negative insulin-like growth factor-I receptor (KR-IGF-IR) specifically targeted to the skeletal muscle; qPCR, quantitative real time PCR; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; UPR, unfolded protein response; WT, wild-type; EF, error factor; PDI, protein-disulfide isomerase; GCOS, Affymetrix GeneChip Operating Software; ID, identity; EIF, eukaryotic initiation factor; UFM1, ubiquitin-fold modifier 1; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; ISG, insulin secretory granule; SYTL4, synaptotagmin-like protein-4/granuphilin; PCX, pyruvate carboxylase; ETF, electron transfer flavin protein; PPIB, peptidyl-prolyl cis-trans isomerase B; VCP, valosin-containing protein; CPE, carboxypeptidase E; UQCRH, ubiquinol-cytochrome c reductase complex; EGF, epidermal growth factor; ARMET, Arginine-rich, mutated in early stage tumors. is reaching epidemic proportions and presents a severe health burden worldwide (1Tiffin N. Adie E. Turner F. Brunner H.G. van Driel M.A. Oti M. Lopez-Bigas N. Ouzounis C. Perez-Iratxeta C. Andrade-Navarro M.A. Adeyemo A. Patti M.E. Semple C.A. Hide W. Computational disease gene identification: a concert of methods prioritizes type 2 diabetes and obesity candidate genes.Nucleic Acids Res. 2006; 34: 3067-3081Crossref PubMed Scopus (0) Google Scholar, 2Zimmet P. Alberti K.G. Shaw J. Global and societal implications of the diabetes epidemic.Nature. 2001; 414: 782-787Crossref PubMed Scopus (4276) Google Scholar). The pathogenesis of T2D is thought to be complicated, involving multiple genetic, metabolic, and environmental factors. In high risk T2D subjects, the earliest detectable abnormality is insulin resistance in the skeletal muscle (3De Fronzo R.A. Pathogenesis of type 2 diabetes: metabolic and molecular implications for identifying diabetes genes.Diabetes Rev. 1997; 5: 177-285Google Scholar, 4Eriksson J. Franssila-Kallunki A. Ekstrand A. Saloranta C. Widen E. Schalin C. Groop L. Early metabolic defects in persons at increased risk for non-insulin-dependent diabetes mellitus.N. Engl. J. Med. 1989; 321: 337-343Crossref PubMed Google Scholar, 5Kahn C.R. Vicent D. Doria A. Genetics of non-insulin-dependent (type-II) diabetes mellitus.Annu. Rev. Med. 1996; 47: 509-531Crossref PubMed Scopus (201) Google Scholar), which is characterized by impaired insulin-mediated signaling, glucose usage, gene expression, and glycogen synthesis as well as the accumulation of intramyocellular triglycerides (4Eriksson J. Franssila-Kallunki A. Ekstrand A. Saloranta C. Widen E. Schalin C. Groop L. Early metabolic defects in persons at increased risk for non-insulin-dependent diabetes mellitus.N. Engl. J. Med. 1989; 321: 337-343Crossref PubMed Google Scholar, 6Ducluzeau P.H. Perretti N. Laville M. Andreelli F. Vega N. Riou J.P. Vidal H. Regulation by insulin of gene expression in human skeletal muscle and adipose tissue. Evidence for specific defects in type 2 diabetes.Diabetes. 2001; 50: 1134-1142Crossref PubMed Google Scholar, 7Cusi K. Maezono K. Osman A. Pendergrass M. Patti M.E. Pratipanawatr T. DeFronzo R.A. Kahn C.R. Mandarino L.J. Insulin resistance differentially affects the PI 3-kinase- and MAP kinase-mediated signaling in human muscle.J. Clin. Investig. 2000; 105: 311-320Crossref PubMed Google Scholar, 8Vaag A. Henriksen J.E. Beck-Nielsen H. Decreased insulin activation of glycogen synthase in skeletal muscles in young nonobese Caucasian first-degree relatives of patients with non-insulin-dependent diabetes mellitus.J. Clin. Investig. 1992; 89: 782-788Crossref PubMed Google Scholar, 9Jacob S. Machann J. Rett K. Brechtel K. Volk A. Renn W. Maerker E. Matthaei S. Schick F. Claussen C.D. Haring H.U. Association of increased intramyocellular lipid content with insulin resistance in lean nondiabetic offspring of type 2 diabetic subjects.Diabetes. 1999; 48: 1113-1119Crossref PubMed Scopus (524) Google Scholar, 10Patti M.E. Butte A.J. Crunkhorn S. Cusi K. Berria R. Kashyap S. Miyazaki Y. Kohane I. Costello M. Saccone R. Landaker E.J. Goldfine A.B. Mun E. DeFronzo R. Finlayson J. Kahn C.R. Mandarino L.J. Coordinated reduction of genes of oxidative metabolism in humans with insulin resistance and diabetes: potential role of PGC1 and NRF1.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8466-8471Crossref PubMed Scopus (1512) Google Scholar). However, not all individuals with insulin resistance develop T2D, and it is now clear that T2D only ensues when insulin-producing pancreatic β-cells fail to compensate for the increased metabolic demands associated with insulin resistance (11Porte Jr., D. Clinical importance of insulin secretion and its interaction with insulin resistance in the treatment of type 2 diabetes mellitus and its complications.Diabetes Metab. Res. Rev. 2001; 17: 181-188Crossref PubMed Scopus (102) Google Scholar, 12Leahy J.L. Pathogenesis of type 2 diabetes mellitus.Arch. Med. Res. 2005; 36: 197-209Crossref PubMed Scopus (323) Google Scholar, 13Kahn B.B. Type 2 diabetes: when insulin secretion fails to compensate for insulin resistance.Cell. 1998; 92: 593-596Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). The exact mechanisms underlying β-cell failure associated with most human forms of T2D remain to be identified (14Kloppel G. Lohr M. Habich K. Oberholzer M. Heitz P.U. Islet pathology and the pathogenesis of type 1 and type 2 diabetes mellitus revisited.Surv. Synth. Pathol. Res. 1985; 4: 110-125PubMed Google Scholar, 15Gunton J.E. Kulkarni R.N. Yim S. Okada T. Hawthorne W.J. Tseng Y.H. Roberson R.S. Ricordi C. O'Connell P.J. Gonzalez F.J. Kahn C.R. Loss of ARNT/HIF1β mediates altered gene expression and pancreatic-islet dysfunction in human type 2 diabetes.Cell. 2005; 122: 337-349Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). Several relatively rare monogenic forms of diabetes characterized by defects in β-cells have been identified (5Kahn C.R. Vicent D. Doria A. Genetics of non-insulin-dependent (type-II) diabetes mellitus.Annu. Rev. Med. 1996; 47: 509-531Crossref PubMed Scopus (201) Google Scholar, 15Gunton J.E. Kulkarni R.N. Yim S. Okada T. Hawthorne W.J. Tseng Y.H. Roberson R.S. Ricordi C. O'Connell P.J. Gonzalez F.J. Kahn C.R. Loss of ARNT/HIF1β mediates altered gene expression and pancreatic-islet dysfunction in human type 2 diabetes.Cell. 2005; 122: 337-349Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar, 16Fajans S.S. Bell G.I. Polonsky K.S. Molecular mechanisms and clinical pathophysiology of maturity-onset diabetes of the young.N. Engl. J. Med. 2001; 345: 971-980Crossref PubMed Scopus (798) Google Scholar, 17Mauvais-Jarvis F. Kahn C.R. Understanding the pathogenesis and treatment of insulin resistance and type 2 diabetes mellitus: what can we learn from transgenic and knockout mice?.Diabetes Metab. 2000; 26: 433-448PubMed Google Scholar, 18Nandi A. Kitamura Y. Kahn C.R. Accili D. Mouse models of insulin resistance.Physiol. Rev. 2004; 84: 623-647Crossref PubMed Scopus (196) Google Scholar, 19Scheuner D. Vander M.D. Song B. Flamez D. Creemers J.W. Tsukamoto K. Ribick M. Schuit F.C. Kaufman R.J. Control of mRNA translation preserves endoplasmic reticulum function in beta cells and maintains glucose homeostasis.Nat. Med. 2005; 11: 757-764Crossref PubMed Scopus (284) Google Scholar, 20Stride A. Hattersley A.T. Different genes, different diabetes: lessons from maturity-onset diabetes of the young.Ann. Med. 2002; 34: 207-216Crossref PubMed Google Scholar). In these forms, diabetes is caused by disruptions or mutations in transcription factors that regulate gene expression in β-cells, leading to an early onset of the disease. However, in most cases of T2D, it is predicted that more subtle alterations in multiple genes and proteins that control glucose-stimulated insulin secretion (GSIS) and β-cell survival play a prominent role in determining susceptibility (21Parikh H. Groop L. Candidate genes for type 2 diabetes.Rev. Endocr. Metab. Disord. 2004; 5: 151-176Crossref PubMed Scopus (0) Google Scholar). Thus, in many forms of T2D it is predicted that multiple genes interact to diminish the ability of β-cells to respond to changes in metabolic demand. The potential importance of such complex gene-gene interactions in controlling β-cell function has been supported by several polygenic mutations in animal models (22Shih D.Q. Heimesaat M. Kuwajima S. Stein R. Wright C.V. Stoffel M. Profound defects in pancreatic β-cell function in mice with combined heterozygous mutations in Pdx-1, Hnf-1α, and Hnf-3β.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3818-3823Crossref PubMed Scopus (0) Google Scholar, 23Withers D.J. Burks D.J. Towery H.H. Altamuro S.L. Flint C.L. White M.F. Irs-2 coordinates Igf-1 receptor-mediated β-cell development and peripheral insulin signalling.Nat. Genet. 1999; 23: 32-40Crossref PubMed Scopus (467) Google Scholar, 24Kido Y. Burks D.J. Withers D. Bruning J.C. Kahn C.R. White M.F. Accili D. Tissue-specific insulin resistance in mice with mutations in the insulin receptor, IRS-1, and IRS-2.J. Clin. Investig. 2000; 105: 199-205Crossref PubMed Google Scholar, 25Bruning J.C. Winnay J. Bonner-Weir S. Taylor S.I. Accili D. Kahn C.R. Development of a novel polygenic model of NIDDM in mice heterozygous for IR and IRS-1 null alleles.Cell. 1997; 88: 561-572Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar). A substantial effort toward understanding insulin resistance and β-cell dysfunction in T2D has been made using animal models and human subjects (5Kahn C.R. Vicent D. Doria A. Genetics of non-insulin-dependent (type-II) diabetes mellitus.Annu. Rev. Med. 1996; 47: 509-531Crossref PubMed Scopus (201) Google Scholar, 15Gunton J.E. Kulkarni R.N. Yim S. Okada T. Hawthorne W.J. Tseng Y.H. Roberson R.S. Ricordi C. O'Connell P.J. Gonzalez F.J. Kahn C.R. Loss of ARNT/HIF1β mediates altered gene expression and pancreatic-islet dysfunction in human type 2 diabetes.Cell. 2005; 122: 337-349Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar, 16Fajans S.S. Bell G.I. Polonsky K.S. Molecular mechanisms and clinical pathophysiology of maturity-onset diabetes of the young.N. Engl. J. Med. 2001; 345: 971-980Crossref PubMed Scopus (798) Google Scholar, 17Mauvais-Jarvis F. Kahn C.R. Understanding the pathogenesis and treatment of insulin resistance and type 2 diabetes mellitus: what can we learn from transgenic and knockout mice?.Diabetes Metab. 2000; 26: 433-448PubMed Google Scholar, 18Nandi A. Kitamura Y. Kahn C.R. Accili D. Mouse models of insulin resistance.Physiol. Rev. 2004; 84: 623-647Crossref PubMed Scopus (196) Google Scholar, 19Scheuner D. Vander M.D. Song B. Flamez D. Creemers J.W. Tsukamoto K. Ribick M. Schuit F.C. Kaufman R.J. Control of mRNA translation preserves endoplasmic reticulum function in beta cells and maintains glucose homeostasis.Nat. Med. 2005; 11: 757-764Crossref PubMed Scopus (284) Google Scholar, 20Stride A. Hattersley A.T. Different genes, different diabetes: lessons from maturity-onset diabetes of the young.Ann. Med. 2002; 34: 207-216Crossref PubMed Google Scholar, 26Brunham L.R. Kruit J.K. Pape T.D. Timmins J.M. Reuwer A.Q. Vasanji Z. Marsh B.J. Rodrigues B. Johnson J.D. Parks J.S. Verchere C.B. Hayden M.R. β-Cell ABCA1 influences insulin secretion, glucose homeostasis and response to thiazolidinedione treatment.Nat. Med. 2007; 13: 340-347Crossref PubMed Scopus (295) Google Scholar, 27Petersen K.F. Dufour S. Befroy D. Garcia R. Shulman G.I. Impaired mitochondrial activity in the insulin-resistant offspring of patients with type 2 diabetes.N. Engl. J. Med. 2004; 350: 664-671Crossref PubMed Scopus (1610) Google Scholar, 28Lauro D. Kido Y. Castle A.L. Zarnowski M.J. Hayashi H. Ebina Y. Accili D. Impaired glucose tolerance in mice with a targeted impairment of insulin action in muscle and adipose tissue.Nat. Genet. 1998; 20: 294-298Crossref PubMed Scopus (0) Google Scholar, 29McIntyre E.A. Walker M. Genetics of type 2 diabetes and insulin resistance: knowledge from human studies.Clin. Endocrinol. (Oxf.). 2002; 57: 303-311Crossref PubMed Scopus (42) Google Scholar, 30Prentki M. Nolan C.J. Islet β cell failure in type 2 diabetes.J. Clin. Investig. 2006; 116: 1802-1812Crossref PubMed Scopus (1171) Google Scholar). To this end, DNA microarray analyses provide a powerful tool to search for clues regarding the molecular mechanisms associated with the pathogenesis of T2D. Gene array studies have been performed on islets to study aspects of T2D in animal models (31Garnett K.E. Chapman P. Chambers J.A. Waddell I.D. Boam D.S. Differential gene expression between Zucker Fatty rats and Zucker Diabetic Fatty rats: a potential role for the immediate-early gene Egr-1 in regulation of beta cell proliferation.J. Mol. Endocrinol. 2005; 35: 13-25Crossref PubMed Scopus (0) Google Scholar, 32Ghanaat-Pour H. Huang Z. Lehtihet M. Sjoholm A. Global expression profiling of glucose-regulated genes in pancreatic islets of spontaneously diabetic Goto-Kakizaki rats.J. Mol. Endocrinol. 2007; 39: 135-150Crossref PubMed Scopus (0) Google Scholar, 33Homo-Delarche F. Calderari S. Irminger J.C. Gangnerau M.N. Coulaud J. Rickenbach K. Dolz M. Halban P. Portha B. Serradas P. Islet inflammation and fibrosis in a spontaneous model of type 2 diabetes, the GK rat.Diabetes. 2006; 55: 1625-1633Crossref PubMed Scopus (140) Google Scholar); however, it is ultimately changes at the protein level that affect cellular function. To date, limited information is available concerning large scale dynamic protein expression changes in pancreatic islets linked to this disease. Using two-dimensional gel electrophoresis combined with mass spectrometry, Sanchez et al. (34Sanchez J.C. Converset V. Nolan A. Schmid G. Wang S. Heller M. Sennitt M.V. Hochstrasser D.F. Cawthorne M.A. Effect of rosiglitazone on the differential expression of diabetes-associated proteins in pancreatic islets of C57Bl/6 lep/lep mice.Mol. Cell. Proteomics. 2002; 1: 509-516Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) reported nine differentially expressed proteins between ob/ob (diabetic) and lean mouse islets. Qiu et al. (35Qiu L. List E.O. Kopchick J.J. Differentially expressed proteins in the pancreas of diet-induced diabetic mice.Mol. Cell. Proteomics. 2005; 4: 1311-1318Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar) found three differentially expressed proteins associated with the high fat diet-induced T2D using mouse pancreatic lysates. To further understand the link between insulin resistance and β-cell dysfunction in T2D, we have studied a mouse model of insulin resistance that progressively develops diabetes (36Fernandez A.M. Kim J.K. Yakar S. Dupont J. Hernandez-Sanchez C. Castle A.L. Filmore J. Shulman G.I. Le Roith D. Functional inactivation of the IGF-I and insulin receptors in skeletal muscle causes type 2 diabetes.Genes Dev. 2001; 15: 1926-1934Crossref PubMed Scopus (272) Google Scholar). One unique feature of the MKR mouse is that it does not harbor a genetic defect in β-cells but rather has a dominant-negative insulin-like growth factor-I receptor mutation specifically targeted to the skeletal muscle (36Fernandez A.M. Kim J.K. Yakar S. Dupont J. Hernandez-Sanchez C. Castle A.L. Filmore J. Shulman G.I. Le Roith D. Functional inactivation of the IGF-I and insulin receptors in skeletal muscle causes type 2 diabetes.Genes Dev. 2001; 15: 1926-1934Crossref PubMed Scopus (272) Google Scholar). Our previous studies revealed that the mutation induces a progressive systemic insulin resistance that leads to compensatory increases in islet and β-cell mass, defective GSIS, β-cell dysfunction, and T2D by 8 weeks of age (36Fernandez A.M. Kim J.K. Yakar S. Dupont J. Hernandez-Sanchez C. Castle A.L. Filmore J. Shulman G.I. Le Roith D. Functional inactivation of the IGF-I and insulin receptors in skeletal muscle causes type 2 diabetes.Genes Dev. 2001; 15: 1926-1934Crossref PubMed Scopus (272) Google Scholar, 37Heron-Milhavet L. Haluzik M. Yakar S. Gavrilova O. Pack S. Jou W.C. Ibrahimi A. Kim H. Hunt D. Yau D. Asghar Z. Joseph J. Wheeler M.B. Abumrad N.A. Leroith D. Muscle-specific overexpression of CD36 reverses the insulin resistance and diabetes of MKR mice.Endocrinology. 2004; 145: 4667-4676Crossref PubMed Scopus (44) Google Scholar, 38Asghar Z. Yau D. Chan F. Leroith D. Chan C.B. Wheeler M.B. Insulin resistance causes increased beta-cell mass but defective glucose-stimulated insulin secretion in a murine model of type 2 diabetes.Diabetologia. 2006; 49: 90-99Crossref PubMed Scopus (53) Google Scholar). Therefore, examining the protein expression pattern of diseased MKR compared with healthy islets may provide important clues to the molecular events associated with the dynamic transition of β-cell dysfunction to failure. In this study, we characterized T2D MKR islets by applying an integrated quantitative iTRAQ proteomics and DNA microarray approach combined with Western blot and quantitative real time PCR for validation. A total of 159 proteins were dysregulated in diabetic MKR islets compared with controls. Functional cluster analysis of these proteins revealed a marked up-regulation of protein biosynthesis and endoplasmic reticulum (ER) stress pathways and a concomitant down-regulation in insulin processing and secretion, as well as mitochondrial energy metabolism pathways in MKR islets. In addition to the affirmation of known diabetogenic proteins, this study revealed novel proteins involved in ER stress and mitochondrial oxidative metabolism that may be associated with β-cell dysfunction and T2D. Mice were maintained in a standard 12-h light/dark cycle and had free access to water and food (diet number 8664; Harlan Tekland, Madison, WI). MKR mice were genotyped by PCR analysis of tail DNA (37Heron-Milhavet L. Haluzik M. Yakar S. Gavrilova O. Pack S. Jou W.C. Ibrahimi A. Kim H. Hunt D. Yau D. Asghar Z. Joseph J. Wheeler M.B. Abumrad N.A. Leroith D. Muscle-specific overexpression of CD36 reverses the insulin resistance and diabetes of MKR mice.Endocrinology. 2004; 145: 4667-4676Crossref PubMed Scopus (44) Google Scholar). All studies were performed on male mice. Wild-type (WT) FVB mice (Charles River, Wilmington, MA) were used as controls. Animal care procedures were conducted according to protocols and the standards of the Canadian Council on Animal Care and approved by the Animal Care and Use Committee at the University of Toronto. Mouse blood glucose and insulin levels were measured from tail vein blood using a glucometer (Bayer, Toronto, Ontario, Canada) and radioimmunoassays (Linco Research, St. Charles, MO), respectively, under non-fasting conditions (39Joseph J.W. Koshkin V. Zhang C.Y. Wang J. Lowell B.B. Chan C.B. Wheeler M.B. Uncoupling protein 2 knockout mice have enhanced insulin secretory capacity after a high-fat diet.Diabetes. 2002; 51: 3211-3219Crossref PubMed Google Scholar). Pancreatic islets were isolated from age-matched male MKR and WT mice by collagenase digestion as described previously (39Joseph J.W. Koshkin V. Zhang C.Y. Wang J. Lowell B.B. Chan C.B. Wheeler M.B. Uncoupling protein 2 knockout mice have enhanced insulin secretory capacity after a high-fat diet.Diabetes. 2002; 51: 3211-3219Crossref PubMed Google Scholar). Briefly the pancreatic duct was perfused with 3 ml of type V collagenase (Sigma). The pancreas was then dissected and digested by incubating for 13–15 min at 37 °C in 5 ml of type V collagenase. Islets were hand-picked three times under a dissecting microscope to remove as much exocrine tissue contamination as possible. Islets were either cultured in RPMI 1640 medium (containing 10% fetal bovine serum and 11.1 mm glucose) or processed for protein and RNA preparation immediately. Pancreatic islet morphology was determined in 3- and 10-week-old mice. Mice were sacrificed, and the pancreas was removed, fixed in 4% paraformaldehyde before being mounted in paraffin blocks, and sectioned for immunostaining with an insulin antibody as described previously (40Kim H. Haluzik M. Asghar Z. Yau D. Joseph J.W. Fernandez A.M. Reitman M.L. Yakar S. Stannard B. Heron-Milhavet L. Wheeler M.B. Leroith D. Peroxisome proliferator-activated receptor-α agonist treatment in a transgenic model of type 2 diabetes reverses the lipotoxic state and improves glucose homeostasis.Diabetes. 2003; 52: 1770-1778Crossref PubMed Google Scholar). Images of freshly isolated islets were taken using a Zeiss LSM510 laser scanning microscope. Glucose-stimulated insulin secretion studies were performed in 3- and 10-week-old mice as reported previously (38Asghar Z. Yau D. Chan F. Leroith D. Chan C.B. Wheeler M.B. Insulin resistance causes increased beta-cell mass but defective glucose-stimulated insulin secretion in a murine model of type 2 diabetes.Diabetologia. 2006; 49: 90-99Crossref PubMed Scopus (53) Google Scholar, 40Kim H. Haluzik M. Asghar Z. Yau D. Joseph J.W. Fernandez A.M. Reitman M.L. Yakar S. Stannard B. Heron-Milhavet L. Wheeler M.B. Leroith D. Peroxisome proliferator-activated receptor-α agonist treatment in a transgenic model of type 2 diabetes reverses the lipotoxic state and improves glucose homeostasis.Diabetes. 2003; 52: 1770-1778Crossref PubMed Google Scholar). The isolated islets were hand-picked and cultured overnight prior to glucose stimulation. Insulin secretion was measured from groups of 20 islets using Krebs-Ringer bicarbonate buffer solution containing 2.8 or 20 mm glucose. Islet DNA was extracted using acid-ethanol, and DNA was calculated for normalization. Insulin concentrations were measured by radioimmunoassays. An overview of the iTRAQ work flow is shown in Fig. 2A. For a complete description of the iTRAQ labeling reaction and the methods for analyses please refer to Refs. 41Unwin R.D. Pierce A. Watson R.B. Sternberg D.W. Whetton A.D. Quantitative proteomic analysis using isobaric protein tags enables rapid comparison of changes in transcript and protein levels in transformed cells.Mol. Cell. Proteomics. 2005; 4: 924-935Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar and 42Shilov I.V. Seymour S.L. Patel A.A. Loboda A. Tang W.H. Keating S.P. Hunter C.L. Nuwaysir L.M. Schaeffer D.A. The Paragon Algorithm, a next generation search engine that uses sequence temperature values and feature probabilities to identify peptides from tandem mass spectra.Mol. Cell. Proteomics. 2007; 6: 1638-1655Abstract Full Text Full Text PDF PubMed Scopus (943) Google Scholar. To decrease eventual biases caused by biological variations, islets from 8–10 mice of each group were pooled yielding one sample. Islet protein samples were prepared in cell lysis buffer (Cell Signaling Technology Inc., Beverly, MA) and kept in liquid N2 until use in the proteomics study. The protein concentration was measured using a BCA™ protein assay kit (Pierce). Three iTRAQ analyses using three independently isolated pancreatic islet samples each containing islets from 8–10 mice were performed. The iTRAQ reagent labeling was performed according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA). Before performing islet sample labeling, a defined six-protein mixture (Applied Biosystems) was labeled and used to confirm the accuracy of ratiometric quantitation of the iTRAQ reagents. Islet protein lysates from MKR and WT mice were purified by acetone precipitation. 150 μg of islet protein from each group was dissolved in 20 μl of dissolution buffer and 1 μl of denaturant reagent. The samples were reduced by addition of 2 μl of reducing reagent and incubation at 60 °C for 1 h. Reduced cysteine residues were then blocked by addition of 1 μl of cysteine blocking reagent and incubated at room temperature for a further 10 min. Tryptic digestion was initiated by the addition of 10 μl of trypsin solution (Applied Biosystems; prepared as 0.5 μg/μl in water solution with enzyme and substrate ratio of ∼1:30) and incubated at 37 °C for 12–16 h. To label the peptides wi
Referência(s)